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Expression of Androgen Receptor Coregulators in Prostate Cancer

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Expression of Androgen Receptor Coregulators in Prostate Cancer 1032 Vol. 10, 1032–1040, February 1, 2004 Clinical Cancer Research Expression of Androgen Receptor Coregulators in Prostate Cancer Marika J. Linja,1 Kati P. Porkka,1 Conclusions: These findings suggest that the decreased Zhikang Kang,3 Kimmo J. Savinainen,1 expression of PIAS1 and SRC1 could be involved in the progression of prostate cancer. In addition, gene amplifica- Olli A. Ja¨nne,3 Teuvo L. J. Tammela,2 4 3 tion of SRC1 in one of the xenografts implies that, in some Robert L. Vessella, Jorma J. Palvimo, and tumors, genetic alteration of SRC1 may provide a growth 1 Tapio Visakorpi advantage. 1Laboratory of Cancer Genetics, Institute of Medical Technology and 2Department of Urology, University of Tampere and Tampere University Hospital, Tampere, Finland; 3Institute of Biomedicine, INTRODUCTION 4 University of Helsinki, Helsinki, Finland; and Department of The critical role of androgens in the development of pros- Urology, University of Washington, Seattle, Washington tate cancer is indicated, for example, by the fact that prostate cancer does not develop in men castrated early in their life (1). ABSTRACT In addition, more that 50 years ago, Huggins and Hodges (2) Purpose: The androgen receptor (AR)-mediated signal- showed that hormonal therapy is an effective treatment for ing pathway seems to be essentially involved in the develop- prostate cancer. Subsequently, androgen withdrawal has become ment and progression of prostate cancer. In vitro studies the standard and is practically the only effective treatment for have shown that altered expression of AR coregulators may advanced prostate cancer. Although most prostate carcinomas significantly modify transcriptional activity of AR, suggest- are originally androgen dependent, they eventually become hor- ing that these coregulators could also contribute to the mone refractory during treatment (3). The mechanisms under- progression of prostate cancer. Here, our goal was to assess lying the transition from androgen dependence to androgen alterations in the expression of the AR coregulators in pros- independence are incompletely understood. tate cancer in vivo. Androgen action in target tissues, such as the prostate Experimental Design: The expression of 16 AR coacti- gland, is mediated by nuclear androgen receptor (AR), which vators and corepressors (SRC1, ␤-catenin, TIF2, PIAS1, functions as a transcription factor. Several findings have already PIASx, ARIP4, BRCA1, AIB1, AIB3, CBP, STAT1, NCoR1, indicated that AR is involved in the development and progres- AES, cyclin D1, p300, and ARA24) was measured in prostate sion of prostate cancer. First, it has been suggested that certain cancer cell lines, xenografts, and clinical prostate tumor AR polymorphisms are associated with the risk of prostate specimens by using real-time quantitative reverse transcrip- cancer (4, 5). Second, somatic mutations of the AR gene have tion-PCR. In addition, gene copy number of SRC1 was been found, especially in tumors treated with antiandrogens analyzed by fluorescence in situ hybridization. such as flutamide and bicalutamide (6–8). Third, about one- Results: Both AR-positive and AR-negative cell lines third of the hormone-refractory prostate carcinomas contain and xenografts expressed the coregulators. Most of the co- amplification of the AR gene, leading to transcriptional up- regulators studied were expressed at equal levels in benign regulation of the gene (9, 10). In addition, hormone-refractory prostatic hyperplasia and untreated and hormone-refrac- tumors express more AR than the untreated tumors, even without tory carcinomas. However, the expression of PIAS1 and the gene amplification (10). Fourth, other growth factor signal- and 0.017, respectively) ing pathways may activate AR, especially in the presence of 0.048 ؍ SRC1 was significantly (P lower in hormone-refractory prostate tumors than in un- only low levels of androgens (11, 12). Finally, recent cDNA treated prostate tumors. No overexpression of the coregula- microarray studies (13, 14) have demonstrated that many of the tors was found in the clinical material. Paradoxically, the androgen-regulated genes become up-regulated at the progres- SRC1 gene was found to be amplified and highly expressed sion of the disease during androgen withdrawal. in a LuCaP 70 prostate cancer xenograft. Activation of AR by androgens is a complex process. The apo-AR stays associated with chaperone proteins in the cyto- plasm. Ligand binding leads to conformational changes in the receptor and its translocation into the nucleus, where it binds to an androgen response element located in the regulatory regions Received 6/29/03; revised 11/4/03; accepted 11/4/03. of target genes (15). In addition to the AR itself, transcriptional Grant support: The Academy of Finland, the Cancer Society of Fin- regulation involves a large number of activating and repressing land, the Reino Lahtikari Foundation, the Medical Research Fund of proteins (16). In vitro studies have indicated that altered expres- Tampere University Hospital, Biocentrum Helsinki, the Sigrid Juse´lius Foundation, and the Finnish Life and Pension Insurance Companies. sion of some of these coregulators may modify transcriptional The costs of publication of this article were defrayed in part by the activity of AR (17–23). For example, overexpression of a p160 payment of page charges. This article must therefore be hereby marked family member, TIF2, in a cotransfection assay enhanced AR advertisement in accordance with 18 U.S.C. Section 1734 solely to transcriptional activity in the presence of several steroids, in- indicate this fact. Requests for reprints: Tapio Visakorpi, Institute of Medical Technol- cluding adrenal androgens, within physiological concentrations ogy, FIN-33014 University of Tampere, Tampere, Finland. Phone: (23). In addition, coregulators may modulate the effects of 358-3-215-7725; Fax: 358-3-215-8597; E-mail: [email protected]. antiandrogens. For instance, it has been reported that hydroxy- Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 1033 flutamide paradoxically increases AR activity when CBP is standard curves, total RNA from LNCaP was used. After first- overexpressed (24). Therefore, changes in the expression of the strand cDNA synthesis, serial dilutions were made correspond- coregulators might be involved in the development and progres- ing to about 100, 20, 4, 0.8, 0.16, and 0.032 ng of total RNA. sion of prostate cancer (25). However, only a few studies on the Primer sequences used for PCR amplification of each gene are expression of the coregulators in prostate cancer have been given in Table 1. The primers were designed to correspond to published (22, 23, 26–28). different exons to avoid amplification of genomic DNA. We To evaluate the significance of alterations in the expression also checked carefully to ensure that the primers could not of AR coregulators in prostate cancer, we have measured the detect pseudogenes or retropseudogenes. The PCR reactions expression of 16 different putative AR coactivators and core- were performed in LightCycler apparatus (29) using an LC Fast pressors by using quantitative reverse transcription-PCR (RT- Start DNA SYBR Green I Kit (Roche Diagnostics, Mannheim, PCR). Clinical tumor samples, prostate cancer cell lines, and Germany). Thermocycling for each reaction was done in a final xenografts were analyzed. The expression data were also com- volume of 20 ␮l containing 2 ␮l of cDNA sample (or standard), ␮ ϫ bined with the data on the chromosomal alterations of the cell 4mM MgCl2, 0.5 M each primer, and 1 ready-to-use SYBR lines and xenografts, obtained previously by comparative Green I reaction mix including Taq DNA polymerase, reaction genomic hybridization, to identify putative amplifications of the buffer, and deoxynucleotide triphosphate mix. The cycling con- coregulator genes. Finally, the gene copy number of SRC1 was ditions were designed according to the manufacturer’s guide- studied by using fluorescence in situ hybridization (FISH). lines. The annealing temperatures are given in Table 1. The elongation time was calculated by dividing the size of the amplicon (Table 1) by 25. The fluorescence emitted by SYBR MATERIALS AND METHODS Green I was measured in every cycle at the end of the elongation Samples. The prostate cancer cell lines (LNCaP, PC-3, step or, to avoid any fluorescence from nonspecific products, at DU145, 22Rv1, and NCI-H660) were obtained from the Amer- higher temperatures (for PIASx, CBP, cyclin D1, p300, and ican Type Culture Collection (Manassas, VA) and cultured ARA24). After arithmetic background adjustment, the fit point under the recommended conditions. Freshly frozen samples of method was used to determine the crossing-point value as de- 10 prostate cancer xenografts (LuCaP 23.1, 23.8, 23.12, 35, 41, scribed previously (10). Fig. 1 illustrates an example of a 49, 58, 69, 70, and 73) were made available for the analyses by standard curve for PIAS1. For normalization of the expression one of the coauthors (R. L. V.). All xenografts, except LuCaP 49 levels, the expression of TATA box-binding protein (TBP) was and LuCaP 58, have been established from hormone-refractory measured as described previously (10). TBP was chosen as the human prostate carcinomas and propagated in intact male mice. reference gene because there are no known retropseudogenes for Freshly frozen prostate tumor specimens representing benign
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